α-aminoacyl β-aminoacyl aminodiols as anti-hypertensive agents

ABSTRACT

Compounds characterized generally as α-aminoacyl β-aminoactyl aminodiols are useful as renin inhibitors for the treatment of hypertension.

FIELD OF THE INVENTION

Renin-inhibiting compounds are known for control of hypertension. Ofparticular interest herein are peptidyl compounds useful as renininhibiting agents.

BACKGROUND OF THE INVENTION

Renin is a proteolytic enzyme produced and secreted into the bloodstreamby the juxtaglomerular cells of the kidney. In the bloodstream, renincleaves a peptide bond in the serum protein angiotensinogen to produce adecapeptide known as angiotensin I. A second enzyme known as angiotensinconverting enzyme, cleaves angiotensin I to produce the octapeptideknown as angiotensin II. Angiotensin II is a potent pressor agentresponsible for vasoconstriction and elevation of cardiovascularpressure. Attempts have been made to control hypertension by blockingthe action of renin or by blocking the formation of angiotensin II inthe body with inhibitors of angiotensin I converting enzyme.

Classes of compounds published as inhibitors of the action of renin onangiotensinogen include renin antibodies, pepstatin and its analogs,phospholipids, angiotensinogen analogs, pro-renin related analogs andpeptide aldehydes.

A peptide isolated from actinomyces has been reported as an inhibitor ofaspartyl proteases such as pepsin, cathepsin D and renin [Umezawa et al,in J. Antibiot. (Tokyo), 23:259-262 (1970)]. This peptide, known aspepstatin, was found to reduce blood pressure in vivo after the theinjection of hog renin into nephrectomized rats [Gross et al, Science,175, 656 (1971)]. Pepstatin has the disadvantages of low solubility andof inhibiting acid proteases in addition to renin. Modified pepstatinshave been synthesized in an attempt to increase the specificity forhuman renin over other physiologically important enzymes. While somedegree of specificity has been achieved, this approach has led to ratherhigh molecular weight hepta- and octapeptides [Boger, et al, Nature,303, 81 (1983)]; high molecular weight peptides are generally consideredundesirable as drugs because gastrointestinal absorption is impaired andplasma stability is compromised.

Short peptide aldehydes have been reported as renin inhibitors [Kokubuet al, Biochim. Biophys. Res. Commun., 118, 929 (1984); Castro et al,FEBS Lett., 167, 273 (1984)]. Such compounds have a reactive C-terminalaldehyde group and would likely be unstable in vivo.

Other peptidyl compounds have been described as renin inhibitors. EPAppl. No. 128,762, published Dec. 18, 1984, describes dipeptide andtripeptide glycol-containing compounds as renin inhibitors [also seeHanson et al, Biochm. Biophys. Res. Comm., 132:155-161 (1985), 146:959-963 (1987)]. EP Appl. No. 181,110, published May 14, 1986, describesdipeptide histidine derivatives as renin inhibitors. EP Appl. No.189,203, published July 30, 1986, describes peptidylaminodiols as renininhibitors. EP Appl. No. 200,406, published Dec. 10, 1986, describesalkyl naphthylmethyl propionyl-histidyl aminohydroxy alkanoates as renininhibitors.

For other articles describing previous efforts to devise renininhibitors, see Marshall, Federation Proc., 35:2494-2501 (1976); Burtonet al, Proc. Natl. Acad. Sci. USA 77: 5476-5479 (1980); Suketa et al,Biochemistry, 14: 3188 (1975;) Swales, Pharmac. Ther., 7: 173-201(1979); Kokubu et al, Nature, 217: 456-457 (1986); Matsushita et al, J.Antibiotics, 28: 1016-1018 (1975); Lazar et al, Biochem. Pharma., 23:2776-2778 (1974); Miller et al., Biochem. Pharma., 21: 2941-2944 (1972);Haber, Clinical Science, 59: 7s-19s (1980); Rich et al, J. Org. Chem.,3624 (1978); J. Med. Chem., 23: 27 (1980); especially Haber, Clin. andExper. Hyper., A5(7&8), 1193 (1983); and European Patent Applications172346A and 172347A published Feb. 26, 1986.

DESCRIPTION OF THE INVENTION

Peptidyl α-aminoacyl β-aminoacyl aminodiols compounds having utility asrenin inhibitors for treatment of hypertension in mammals constitute afamily of compounds of general Formula I: ##STR1## wherein R₉ isselected from H, lower alkyl, alkoxy, alkylamino, benzyloxycarbonyl,phenyl, phenyl substituted with one or more of halo, methoxy, hydroxy,alkyl, amino, aminoalkyl and trifluoromethyl, ##STR2## wherein Y and Qare selected from CH₂, ##STR3## O, S, SO, SO₂ and NR₁₃, wherein R₁₂ is Hor lower alkyl, R₁₃ is selected from H, phenyl and ##STR4## and whereinR₁₄ is H or lower alkyl; wherein each of m and n is independently aninteger from 1 through 4; wherein each of r, t, u and v is independentlyan integer from zero through 2; wherein p is an integer from 1 through3; wherein each of a through d is independently an integer from zerothrough 3; wherein T is selected from one or more groups selected fromH, linear or branched lower alkyl, alkoxy, oxo, halo, haloalkyl, loweralkenyl, lower alkynyl and cyano; wherein R₁ is selected from H, linearor branched lower alkyl, haloalkyl, alkylcycloalkyl, alkylcycloalkenyland alkoxycarbonyl; wherein R₂ is selected from linear or branched loweralkyl and benzyl and imidazolemethyl; wherein R₃ is selected from loweralkyl, acylaminoalkyl, benzyl, naphthylmethyl, aryl and benzylsubstituted at the phenyl portion by halo or lower alkyl or by both;wherein each of R₄ and R₅ is independently selected from H and loweralkyl; wherein R₆ is H or phenyl; wherein R₇ is selected fromsubstituted or unsubstituted cycloalkyl, phenyl, cycloalkylalkyl andphenylalkyl, any one of which may be substituted with one or more groupsselected from alkyl, alkoxy, halo, haloalkyl, lower alkenyl, loweralkynyl and cyano; and wherein each of R₈ is H or lower alkyl; whereineach of R₁₀ and R₁₁ is independently selected from the groups H, loweralkyl, cycloalkyl, phenyl, benzyl, naphthyl and naphthylmethyl, any oneof which groups having a substitutable position may be optionallysubstituted with or more of lower alkyl, alkoxy, alkenyl, alkynyl, halo,haloalkyl, cyano and phenyl, with the proviso that at least one of R₁₀and R₁₁ is an aryl group.

A preferred group of compounds within Formula I are those compoundshaving the specific stereochemical configuration shown in Formula II##STR5##

Preferred compounds within Formula II are those compounds wherein Y andQ are selected from O, CH₂, S, NR₁₃ wherein R₁₃ is H or ##STR6## whereinR₁₄ is H or lower alkyl; wherein each of m and n is an integer from 1through 3; wherein each of r, t, u and v is independently zero or one;wherein p is one or two; wherein each of a through d is independently aninteger from zero through two; wherein T is selected from one or moregroups selected from H, lower alkyl, oxo and halo; wherein R₁ isselected from H, lower alkyl, alkylcycloalkyl and alkoxycarbonyl;wherein R₂ is selected from lower alkyl imidazolemethyl and benzyl;wherein R₃ is selected from lower alkyl, acylaminoalkyl, benzyl,napthylmetnyl, aryl and benzyl substituted at the phenyl portion by haloor lower alkyl or by both; wherein R₄ is selected from H and loweralkyl; wherein R₅ is H or methyl; R₆ is H or methyl; wherein R₇ isselected from cyclohexylmethyl and benzyl; R₈ is H or lower alkyl;wherein each of R₁₀ and R₁₁ is independently selected from H, phenyl,naphthyl and phenyl substituted with one or more lower alkyl, alkoxy,alkenyl, halo, cyano and phenyl, with the proviso that at least one ofR₁₀ and R₁₁ is phenyl.

Within the aforementioned preferred group of compounds, there are foursub-groups of preferred compounds. The first sub-group consists of thosecompounds of Formula II wherein R₉ is ##STR7## wherein Y is O, CH₂, orS; m is 2; n is 2; T is one or more of H or lower alkyl; R₁ is H ormethyl; or lower alkyl; R₂ is lower alkyl; R₃ is benzyl; R₄ is H; R₅ isH or methyl; R₆ is H or methyl; and R₇ is cyclohexylmethyl. Of thisfirst sub-group the most preferred are those compounds wherein Y is O; mis 2; n is 2; T is one or more of H or methyl; R₁ is selected from H,methyl, ethyl and isobutyl; R₂ is isobutyl; R₃ is selected from benzyland napthylmethyl; R₄ is H or methyl; R₅ is H or methyl; R₆ is H ormethyl; R₇ is cyclohexylmethyl; and R₈ is H or methyl. Radicals whichexemplify the R₉ substituent as shown in Formula III are as follows:##STR8##

Examples of specific compounds within this sub-group are the following:

Boc-L-phenylalaninyl-D,L-α-methyl-β-alaninamide of(2S,3R,4S)-2-amino-1-cyclohexyl-3,4-dihydroxy-6-methylheptane;Boc-L-phenylalaninyl-D,L-α-methyl-β-alaninamide of(2S,3R,4S)-2-amino-1-cyclohexyl-3,4-dihydroxypentane; andBoc-L-Phenylalaninyl-D,L-α-methyl-β-alaninamide of(2S,3R)-2-amino-1-cyclohexyl-3,4-dihydroxybutane.

A second sub-group of preferred compounds consists of those withinFormula I wherein R₉ is ##STR9## wherein Y is selected from O, S, SO,CH₂, and SO₂ ; wherein each of r, t, u and v is independently zero orone; p is 1 or 2; T is one or more of H or lower alkyl; R₁ is loweralkyl; R₂ is lower alkyl; R₃ is benzyl; R₄ is H or methyl; R₅ is H ormethyl; R₆ is H or methyl; R₇ is cyclohexylmethyl; R₈ is H or methyl.The substituent of Formula IV may be substituted at any substitutableposition within the bicyclic structure of Formula IV.

Radicals which exemplify the R₉ substituent of Formula I are as follows:##STR10##

A third sub-group of preferred compounds consists of those compoundswherein R₉ is ##STR11## wherein Q is O, CH₂, or S; a is zero; b is 1; cis 1; d is zero; each T is independently one or more of H or loweralkyl; R₁ is H or lower alkyl; R₂ is lower alkyl; R₃ is benzyl; R₄ is Hor methyl; R₅ is H or methyl; R₆ is H or methyl; and R₇ iscyclohexylmethyl; R₈ is H or methyl.

Within this third sub-group is a set of more preferred compounds ofFormula I wherein Q is O, R₁ is selected from H, methyl, ethyl andisobutyl, and R₂ is isobutyl, an example of which is a compound whereinR₁ is isobutyl, and R₇ is cyclohexylmethyl. Another set of morepreferred compounds within this third sub-group are those wherein Q isS, R₁ is selected from H, methyl and isobutyl, and R₂ is isobutyl, anexample of which is a compound wherein R₁ is isobutyl, and R₇ iscyclohexylmethyl. Radicals which exemplify the R₉ substituent as shownin Formula V: ##STR12##

Within any of these radicals exemplifying Formulae III, IV and V, thesubstituent R represents a linear or branched alkyl group of one toabout ten carbon atoms, or preferably, one to about five carbon atoms.

A fourth sub-group of preferred compounds consists of those compounds ofFormula II wherein R₉ is ##STR13## wherein each of R₁₀ and R₁₁ isindependently selected from the groups H, lower alkyl, cycloalkyl,phenyl, benzyl, naphthyl, and naphthylmethyl, any one of which groupshaving a substitutable position may be optionally substituted with ormore of lower alkyl, alkoxy, alkenyl, alkynyl, halo, haloalkyl, cyanoand phenyl, with the proviso that at least one of R₁₀ and R₁₁ is an arylgroup.

Of this fourth subgroup, more preferred compounds are those wherein R₁is isobutyl; R₂ is isobutyl; R₃ is benzyl; R₄ is H or methyl; R₅ is H ormethyl; R₆ is H or methyl; R₇ is cyclohexylmethyl R₈ is H or methyl; andeach of R₁₀ and R₁₁ is independently selected from H, lower alkyl andphenyl, with at least one of R₁₀ and R₁₁ being phenyl. An especiallypreferred compound is wherein R₁₀ is H and R₁₁ is phenyl.

Unless otherwise described, the chemical groups recited herein shallhave meanings as follows: "Lower alkyl" means alkyl radicals containingone to about 10 carbon atoms in a linear or branched configuration,examples of which include methyl, ethyl, n-propyl, isopropyl, n-butyl,sec-butyl, iso-butyl, tert-butyl, n-pentyl, neopentyl, n-hexyl,1-methylhexyl, n-heptyl, 2-ethylheptyl, n-octyl, 3-propyloctyl, n-nonyl,4-butylnonyl, n-decyl and the like. "Lower alkenyl" and "lower alkynyl"mean, respectively, hydrocarbon radicals of two to about ten carbonatoms containing at least one carbon-carbon double bond and at least onecarbon-carbon triple bond, respectively, attached to alkyl radicals ofthe type embraced by the term "lower alkyl" examples of which are2-butenyl and 2-pentynyl. "Haloalkyl" means alkyl radicals substitutedat one or more substitutable positions with one or more halo groups.Preferred haloalkyl group are those provided by lower alkyl radicalssubstituted at least at one position with one, two or three halo groupssuch as fluoro or chloro, a specific example of which istrifluoromethyl. "Alkylcycloalkyl" means a cyclized alkyl having fromfour to about nine ring carbon atoms, any one or more of thesubstitutable ring carbons being substituted with an alkyl group,preferably a lower alkyl group. "Alkylcycloalkenyl" means a cyclizedhydrocarbon radical containing four to about nine ring carbon atomscontaining at least one carbon-carbon double bond, but less than thenumber of double bonds required to form a fully unsaturated ring system,any one or more of the substitutable ring carbon atoms being substitutedwith an alkyl group, preferably a lower alkyl group. "Alkoxycarbonyl"means an oxycarbonyl radical having an alkyl, preferably lower alkyl,group attached to the oxygen atom. "Aryl" means an aromatic hydrocarbonradical provided by a homocyclic or heterocyclic ring system, such asphenyl, naphthyl, and pyridyl. "Acyl" means a carbonyl radical attachedto an alkyl or aryl group.

Based upon the foregoing, the meanings of the following terms should bereadily discernible, namely, "acylaminoalkyl", "cycloalkyl","cycloalkylalkyl", "phenylalkyl" and "alkoxy".

In the cyclic structures of Formulae III, IV and V where the substituentT is shown, it is intended that the T substituent represents one or moresubstituents which may be attached at any substitutable position on anyof the described cyclic structures.

Compounds of Formula I may have two or more carbon atoms providingasymmetric sites which are important for conferring activity. Preferredcompounds have three asymmetric carbons which lead to confer improvedactivity. Such compounds whether in their pure isomer form or asdiastereomeric mixtures are embraced in the Formula I and II compounds,of the invention. Many of the more active renin inhibitors are providedby compounds having a specific stereochemical configuration. WithinFormula I and II, reading from the N terminus to the C terminus(terminating with the diol moiety), the preferred configurations for theasymmetric carbons are as follows: S, R or S, R or S, S, R, S.

Compounds of Formula I have been found to inhibit the production ofangiotensin II in mammals. Angiotensin II is a potent vasoconstrictorand participates in the formation of aldosterone which regulates sodiumand water balance in mammals. Thus, compounds of Formula I aretherapeutically useful in methods for treating hypertension byadministering to a hypertensive patient a therapeutically-effectiveamount of a compound of Formula I.

These compounds can be formulated into pharmaceutically-acceptabledosage forms by any of a number of well-known carriers or diluents. Thecompounds can be formulated using pharmacologically-acceptable acidaddition salts and can be used in a suitable hydrated form. Theformulated compounds can be administered in oral dosage forms such astablets, capsules, pills, powders, or granules. The compounds can alsobe administered intramuscularly, using forms known to the pharmaceuticalart. In general, the preferred form of administration is oral. Atherapeutically-effective but non-toxic quantity of the compound isemployed in treatment of high blood pressure in mammals. The dosageregimen for preventing or treating hypertension with the compounds ofFormula I is selected upon consideration of a variety of factors,including the type, age, weight, sex, and medical condition of thepatient, the severity of the hypertension, the route of administration,and the particular compound employed. Dosages of the compounds areordinarily in the range from about 0.5 to about 100 mg/kg (activecompound-to-body weight), and preferably from about 1.0 to about 20mg/kg given orally or by injection.

Compounds of Formula I are also useful as diagnostic agents foridentification of hypertension due to renin excess.

Compounds of Formula I can be administered as prodrugs. Preferably,esterification of one or more of the hydroxyl groups of the compounds ofFormula I is accomplished with amino acids to make aminoesters,succinates to make succinic acid esters, or phosphates to makephosphoric acid esters. Aminoesters of the Formula I compounds are morepreferred.

Procedures for preparation of compounds of Formula I are set forth inthe following Synthetic Scheme and Generic Synthetic Description takenwith the specific procedures described in Examples 1-9 which followthereafter. The substituents R₁ through R₉ are as described above forthe Formula I substituents.

SYNTHETIC SCHEME ##STR14## Generic Synthetic Description

An allylic acetate, appropriately substituted and suitably protected, asshown in the scheme is used as starting material. This substance, andlike substances, are ozonized under standard conditions (lowtemperature, as methanol-methylene chloride solutions) and the reductionof the ozonide to an aldehyde is effected with dimethyl sulfide. Onceobtained, this type of aldehyde is treated with organometallic reagentscapable of delivering an alkyl group to the aldehyde to produce diols ofthe type shown. These diols may then be converted, using standardpeptide coupling methodology to renin inhibitors as shown via couplingto the general acid shown in the scheme. The initially obtained diol mayalso be hydrogenated to the saturated cyclohexane diol and again,coupled to in a similar manner to acids of the general description givenin the scheme.

The following examples are provided to illustrate the present inventionand are not intended to limit the scope thereof. Those skilled in theart will readily understand that known variations of the conditions andprocesses of the following preparative procedures can be used to preparethese compounds. All temperatures expressed are in degrees Centigrade.Within the foregoing synthetic description and examples which follow,abbreviations have meansings as indicated below:

BOC=butyloxycarbonyl

i-Bu=isobutyl

Leu=L-leucine

Ac=acyl

Me=methyl

TFA=trifluoroacetic acid

THF=tetrahydrofuran

EXAMPLE 1(3S,4S)-N-[(tert-Butyloxy)carbonyl]-4-amino-3-acetoxy-5-phenylpentene

The preparation of the above intermediate was carried out using theprocedure described in Hanson, et al., (1985) J. Org. Chem. 50,5399.

EXAMPLE 2(2R,3S)-N-[(tert-Butyloxy)carbonyl]-3-amino-2-acetoxy-4-phenylbutanal

The preparation of the above intermediate was carried out as describedin Hanson, et al. above. Ozone/oxygen was bubbled at -70° into asolution of 2.55 g (8.0 mmol) of the allylic acetate of Example 1 in 100mL of methylene chloride until a deep blue color persisted. Oxygen wasintroduced until the blue color completely faded, then 3.0 mL of Me₂ Swas added and the solution was allowed to warm to 0°-5° and standovernight. The solvent was removed at 0° under vacuum yielding the titlecompound as a thick yellow oil which was used in the following stepwithout purification.

EXAMPLE 3(2S,3R,4S)-N-[(tert-Butyloxy)carbonyl]-2-amino-1-phenyl-3,4-dihydroxy-6-methylheptane

The oil prepared in Example 2 was dissolved under nitrogen in 100 mL ofdry THF and cooled to -70°. To this solution was added 13 mL (26 mmol)of a 2.0M solution of isobutylmagnesium chloride in ether and thestirred mixture was allowed to warm to room temperature and stir for 2hrs. After decomposition with MeOH/H₂ O the mixture was diluted withether, washed with saturated NH₄ Cl solution twice, then dried and thesolvents stripped off under vacuum. The residue was allowed to standovernight in 80% MeOH-H₂ O containing excess ammonium hydroxide. TheMeOH was stripped off and the mixture was extracted with ether. Theseextracts were combined, washed with water, dilute KHSO₄ then dried andevaporated to give 2.36 g of a yellow glass which crystallized from 50mL of pentane on standing overnight. The yellow-white powder obtainedwas recrystallized from ether-hexane and furnished the title compound(0.41 g) as white, hairy needles, mp 134°-136°, Rf (ether): single spot,0.6. By chromatography of the mother liquors and crystallization of theappropriate fractions, an additional 0.22 g of product, mp 138°-139° wasobtained. Anal: Calcd. for C₁₉ H₃₁ NO₄ (337.45): C, 67.62; H, 9.26; N,4.15. Found: C, 67.51; H, 9.43; N, 4.24.

EXAMPLE 4(2S,3R,4S)-N-[(tert-Butyloxy)carbonyl]-2-amino-1-cyclohexyl-3,4-dihydroxy-6-methylheptane

The diol of Example 3, 0.27 g, was reduced in MeOH with 60 psi H₂ at 60°in 3 hrs using 5% Rh/C catalyst. After filtering, the solvent wasstripped off and the 0.27 g of white crystals were recrystallized fromCH₂ Cl₂ -hexane to furnish tiny needles of the title compound, 0.19 g,mp 126°-128°; further recrystallization gave mp 128.5°-129.5° Rf(ether): single spot, 0.8.

Anal: Calcd. for C₁₉ H₂₇ NO₄ (343.50): C, 66.43; H, 10.86, N, 4.08.Found: C, 66.43; H, 11.01; N, 4.03.

EXAMPLE 5 Methyl D,L-3-aminoisobutyrate

To a stirred solution of D,L-3-aminoisobutyric acid (5 g) in methanol(150 mL) at 0° was added dropwise thionyl chloride (7.5 mL); the mixturewas allowed to warm to room temperature and stir for 15 hours. Theresulting solution was evaporated to yield the title compound as acrystalline solid: 200 MHz ¹ H NMR: consistent with structure.

EXAMPLE 6 Boc-L-phenylalaninyl-D,L-α-methyl-β-alanine free acid

Boc-L-phenylalanine (6 g) was dissolved in methylene chloride (30 mL)and to this was added N-methylpiperidine (2.38 g). This solution wascooled to -10° and isobutychloroformate (3.3 g) was added, followed by asolution of title compound of Example 5 (3.7 g) and N-methylpiperidine(2.4 g) in methylene chloride after 5 minutes. This mixture was stirredfor 8 hours and evaporated. The residue was taken up in methanol and 1Npotassium hydroxide added. After 5 minutes, 0.5M citric acid was addedand the mixture extracted with ethyl acetate to give the methyl ester ofthe title compound as 1:1 mixture of diastereomers which crystallizesupon standing: Anal. calcd for C₉ H₂₈ N₂ O₅ : C, 62.62; H, 7.75; N,7.68. Found: C: 62.32; H, 7.78; N, 7.66. This ester was saponified with1N sodium hydroxide to yield the title acid as an amorphous solid: Anal.calcd for C₁₈ H₂₆ N₂ O₅ +0.5 H₂ O: C, 60.15; H, 7.57; N, 7.79. Found: C,59.77; H, 7.59; N, 7.22.

EXAMPLE 7 (2S,3R)-N-Boc-2-amino-1-cyclohexyl-3,4-dihydroxybutane

The procedure of Example 4 was employed using(2S,3R)-N-Boc-2-amino-1-phenyl-3,4-dihydroxybutane, prepared by themethod of Hanson, et al., to give crystalline title compound:

Anal. calcd for C₁₅ H₂₉ NO₄ : C, 62.69; H, 10.17; N, 4.87. Found: C,62.69; H, 10.51; N, 4.91.

EXAMPLE 8 Boc-L-phenylalaninyl-D,L-α-methyl-β-alaninamide of(2S,3R)-2-amino-1-cylohexyl-3,4-dihydroxybutane

(2S,3R)-N-Boc-2-amino-1-cyclohexyl-3,4-dihydroxybutane (described inExample 7) was treated with trifluoroacetic acid to remove the Boc groupand the resulting salt was coupled to the title acid of Example 6 usingthe general procedure described in Example 6 for amide formation, toproduce the title renin inhibitor: 400 MHz NMR (CDCl₃): consistent withproposed structure. Anal. calcd for C₂₈ H₄₅ N₃ O₆ +0.5 H₂ O: C, 63 61;H, 8.77; N, 7.94. Found: C, 63.67; H, 8.69; N, 7.80.

EXAMPLE 9 Boc-L-phenylalaninyl-D,L-α-methyl-β-alaninamide of(2S,3R,4S)-2-amino-1-cyclohexyl-3,4-dihydroxy-6-methylheptane

The procedure of Example 8 was used, except that the above diol wasreplaced with(2S,3R,4S)-Boc-2-amino-1-cyclohexyl-3,4-dihydroxy-6-methylheptane togive the title renin inhibitor: 400 MHz NMR (CDCl₃): consistent withproposed structure. Anal. calcd for C₃₂ H₅₃ N₃ O₆ +0.5 H₂ O: C, 65.72;H, 9.31; N, 7.18. Found: 65.73; H, 9.29; N, 6.99.

Biological Evaluation

Compounds of Formula I were evaluated as inhibitors of human and hogrenin in vitro, as follows:

The human renin inhibition test has been previously described in detailin papaioannou, et al. (1985) Clinical and Experimental HypertensionA7(9), 1243-1257. Human renin was obtained from the National Institutefor Biological Standards, London. In a total volume of 0.25 mL 100 mMTris-acetate buffer at pH 7.4, 25 x 10-6 Goldblatt units of renin, 0.05mL of plasma from human volunteers taking oral contraceptives, 6.0 mMsodium EDTA, 2.4 mM phenylmethyl sulfonyl fluoride, 1.5 mM8-hydroxyquinoline, 0.4 mg/mL BSA, and 0.024 mg/mL neomycin sulfate wereincubated for two hours at 37° C. in the presence or absence of renininhibitors. The produced angiotensin I was determined byradioimmunoassay (New England Nuclear kit). Compounds of Formula I to beassayed were solubilized in either ethyl alcohol or DMSO and dilutedwith 100 mM Tris-acetate buffer at pH 7.4 to the appropriateconcentration. The final concentration of organic solvent in thereaction mixture was less than 1%. Control incubations at 37° C. wereused to correct for effects of organic solvent on renin activity.

The hog renin inhibition assay was performed in a manner similar to thehuman renin assay, with the following modifications. Hog renin waspurchased from Sigma Chemical Co. and the synthetic tetradecapeptidesubstrate was obtained from Peninsula Labs Inc. In a final volume of0.25 mL 100 mM Tris-acetate buffer at pH 7.4, 0.125m units hog renin, 20micromolar tetradecapetide, 6 mM disodium EDTA, 3.2 mM phenylmethylsulfonyl fluoride, 3 mM 8-hydroxyquinoline, 1.2 mg/mL BSA and 0.024mg/mL neomycin sulfate were incubated for one hour at 37° C. in thepresence or absence of renin inhibitors. The amount of angiotensin Iproduced was determined as for the human renin inhibition assay.

BIOLOGICAL RESULTS

                  TABLE 1                                                         ______________________________________                                        compound   Human Renin IC.sub.50                                                                         Hog Renin IC.sub.50                                ______________________________________                                        Example 8  2 × 10.sup.-6 M                                                                         2.1 × 10.sup.-6 M                            Example 9  2.1 × 10.sup.-7 M                                                                       1.25 × 10.sup.-8 M                           ______________________________________                                    

Although this invention has been described with respect to specificembodiments, the details of these embodiments are not to be construed aslimitations. Various equivalents, changes and modifications may be madewithout departing from the spirit and scope of this invention, and it isunderstood that such equivalent embodiments are part of this invention.

What is claimed is:
 1. A compound of the formula: ##STR15## wherein R₉is selected from H, alkyl, alkoxy, alkylamino, benzyloxycarbonyl,phenyl, phenyl substititued with one or more of halo, methoxy, hydroxy,amino, aminoalkyl, trifluoromethyl and ##STR16## wherein R₁ is selectedfrom H, linear or branched lower alkyl, haloalkyl, alkylcycloalkyl,alkylcycloalkenyl and alkoxycarbonyl; wherein R₂ is selected from linearor branched lower alkyl, imidazolemethyl and benzyl; wherein R₃ isselected from lower alkyl, loweralkanoylaminoalkyl, benzyl,naphthylmethyl, aryl and benzyl substituted at the phenyl portion byhalo or lower alkyl or by both; wherein each of R₄, R₅, R₆ and R₈ isindependently selected from H or lower alkyl; wherein R₇ is selectedfrom substituted or unsubstituted cycloalkyl, phenyl, cycloalkylalkyland phenylalkyl, any one of which may be substituted with one or moregroups selected from alkyl, alkoxy, halo, haloalkyl, lower alkenyl,lower alkynyl and cyano; and wherein each of R₁₀ and R₁₁ isindependently selected from the groups H, lower alkyl, cycloalkyl,phenyl, benzyl, naphthyl and naphthylmethyl, any one of which groupshaving a substitutable position may be optionally substituted with ormore of lower alkyl, alkoxy, alkenyl, alkynyl, halo haloalkyl, cyano andphenyl, with the proviso that at least one of R₁₀ and R₁₁ is an arylgroup; or a pharmacologically-acceptable salt thereof.
 2. Compound ofclaim 1 of the formula ##STR17## wherein R₁ through R₁₁ are as definedbefore; or a pharmacologically-acceptable salt thereof.
 3. Compound ofclaim 2 which is Boc-L-phenylalaninyl-D,L-α-methyl-β-alaninamide of(2S,3R,4S)-2-amino-1-cyclohexyl-3,4-dihydroxy-6-methylheptane; or apharmacologically-acceptable salt thereof.
 4. Compound of claim 2 whichis Boc-L-phenylalaninyl-D,L-α-methyl-β-alaninamide of(2S,3R,4S)-2-amino-1-cyclohexyl-3,4-dihydroxypentane; or apharmacologically-acceptable salt thereof.
 5. Compound of claim 2 whichis Boc-L-phenylalaninyl-D,L-α-methyl-β-alaninamide of(2S,3R)-2-amino-1-cyclohexyl-3,4-dihydroxybutane; or apharmacologically-acceptable salt thereof.
 6. Compound of claim 2wherein R₉ is ##STR18## wherein R₁ is selected from H, linear orbranched lower alkyl, haloalkyl, alkylcycloalkyl, alkylcycloalkenyl andalkoxycarbonyl; wherein R₂ is selected from linear or branched loweralkyl, imidazolemethyl and benzyl; wherein R₃ is selected from loweralkyl, acylaminoalkyl, benzyl, naphthylmethyl, aryl and benzylsubstituted at the phenyl portion by halo or lower alkyl or by both;wherein each of R₄, R₅, R₆ and R₈ is independently selected from H orlower alkyl; wherein R₇ is selected from substituted or unsubstitutedcycloalkyl, phenyl, cycloalkylalkyl and phenylalkyl, any one of whichmay be substituted with one or more groups selected from alkyl, alkoxy,halo, haloalkyl, lower alkenyl, lower alkynyl and cyano; wherein each ofR₁₀ and R₁₁ is independently selected from the groups H, lower alkyl,cycloalkyl, phenyl, benzyl, naphthyl, and naphthylmethyl, any one ofwhich groups having a substitutable position may be optionallysubstituted with or ore of lower alkyl, alkoxy, alkenyl, alkynyl, halo,haloalkyl, cyano and phenyl, with the proviso that at least one of R₁₀and R₁₁ is an aryl group; or a pharmacologically-acceptable saltthereof.
 7. Compound of claim 6 wherein R₁ is isobutyl; R₂ is isobutyl;R₃ is benzyl; R₄ is H or methyl; R₅ is H or methyl; R₆ is H or methyl;R₇ is cyclohexylmethyl; R₈ is H or methyl; each of R₁₀ and R₁₁ isindependently selected from H, lower alkyl and phenyl, with at least oneof R₁₀ and R₁₁ being phenyl; or a pharmacologically-acceptable saltthereof.
 8. A pharmaceutical composition comprising atherapeutically-effective amount of a resin-inhibiting compound and apharmaceutically-acceptable carrier or diluent, said remain-inhibitingcompound selected from a family of compounds of the formula: ##STR19##wherein R₉ is selected from H, alkyl, alkoxy, alkylamino,benzyloxycarbonyl, phenyl, phenyl substituted with one or more of halo,methoxy, hydroxy, amino, aminoalkyl, trifluoromethyl and ##STR20##wherein R₁ is selected from H, linear or branched lower alkyl,haloalkyl, alkylcycloalkyl, alkylcycloalkenyl and alkoxycarbonyl;wherein R₂ is selected from linear or branched lower alkyl,imidazomethyl and benzyl; wherein R₃ is selected from lower alkyl,loweralkanoylaminoalkyl, benzyl, naphthylmethyl, aryl and benzylsubstituted at the phenyl portion by halo or lower alkyl or by both;wherein each of R₄, R₅, R₆ and R₈ is independently selected from H orlower alkyl; wherein R₇ is selected from substituted or unsubstitutedcycloalkyl, phenyl, cyclolkylalkyl and phenylalkyl, any one of which maybe substituted with one or more groups selected from alkyl, alkoxy,halo, haloalkyl, lower alkenyl, lower alkynyl and cyano; and whereineach of R₁₀ and R₁₁ is independently selected from the groups H, loweralkyl, cycloalkyl, phenyl, benzyl, naphthyl and naphthylmethyl, any oneof which groups having a substitutable position may be optionallysubstituted with or more of lower alkyl, alkoxy, alkenyl, alkynyl, halo,haloalkyl, cyano and phenyl, with the proviso that at least one of R₁₀and R₁₁ is an aryl group; or a pharmacologically-acceptable saltthereof.
 9. The composition of claim 8 wherein said renin-inhibitingcompound is of the formula ##STR21## wherein R₁ through R₁₁ are asdefined before; or a pharmacologically-acceptable salt thereof.
 10. Thecomposition of claim 9 wherein said renin-inhibiting compound isBoc-L-phenylalaninyl-D,L-α-methyl-β-alaninamide of(2S,3R,4S)-2-amino-1-cyclohexyl-3,4-dihydroxy-6-methylheptane; or apharmacologically-acceptable salt thereof.
 11. The composition of claim9 wherein said renin-inhibiting compound isBoc-L-phenylalaninyl-D,L-α-methyl-β-alaninamide of(2S,3R,4S)-2-amino-1-cyclohexyl-3,4-dihydroxypentane; or apharmacologically-acceptable salt thereof.
 12. The composition of claim9 wherein said renin-inhibiting compound isBoc-L-phenylalaninyl-D,L-α-methyl-β-alaninamide of(2S,3R)-2-amino-1-cyclohexyl-3,4-dihydroxybutane; or apharmacologically-acceptable salt thereof.
 13. The composition of claim9 wherein R₉ is ##STR22## wherein R₁ is selected from H, linear orbranched lower alkyl, haloalkyl, alkylcycloalkyl, alkylcycloalkenyl andalkoxycarbonyl; wherein R₂ is selected from linear or branched loweralkyl, imidazolemethyl and benzyl; wherein R₃ is selected from loweralkyl, acylaminoalkyl, benzyl, napthylmethyl, aryl and benzylsubstituted at the phenyl portion by halo or lower alkyl or by both;wherein each of R₄, R₅, R₆ and R₈ is independently selected from H orlower alkyl; wherein R₇ is selected from substituted or unsubstitutedcycloalkyl, phenyl, cycloalkylalkyl and phenylalkyl, any one of whichmay be substituted with one or more groups selected from alkyl, alkoxy,halo, haloalkyl, lower alkenyl, lower alkynyl and cyano; wherein each orR₁₀ and R₁₁ is independently selected from the groups H, lower alkyl,cycloalkyl, phenyl, benzyl, naphthyl, and naphthylmethyl, any one ofwhich groups having a substitutable position may be optionallysubstituted with or more of lower alkyl, alkoxy, alkenyl, alkynyl, halo,haloalkyl, cyano and phenyl, with the proviso that at least one of R₁₀and R₁₁ is an aryl group; or a pharmacologically-acceptable saltthereof.
 14. The composition of claim 13 wherein R₁ is isobutyl; R₂ isisobutyl; R₃ is benzyl; R₄ is H or methyl; R₅ is H or methyl; R₆ is H ormethyl; R₇ is cyclohexylmethyl; R₈ is H or methyl; each of R₁₀ and R₁₁is independently selected from H, lower alkyl and phenyl, with at leastone of R₁₀ and R₁₁ being phenyl; or a pharmacologically-acceptable saltthereof.
 15. A therapeutic method for treating hypertension, said methodcomprising administering to a hypertensive patient atherapeutically-effective amount of a compound of the formula: ##STR23##wherein R₉ is selected from H, alkyl, alkoxy, alkylamino,benzyloxycarbonyl, phenyl, phenyl substituted with one or more of halo,methoxy, hydroxy, amino, aminoalkyl, trifluoromethyl and ##STR24##wherein R₁ is selected from H, linear or branched lower alkyl,haloalkyl, alkylcycloalkyl, alkylcycloalkenyl and alkoxycarbonyl;wherein R₂ is selected from linear or branched lower alkyl,imidazolemethyl and benzyl; wherein R₃ is selected from lower alkyl,loweralkanoylaminoalkyl, benzyl, naphthylmethyl, aryl and benzylsubstituted at the phenyl portion by halo or lower alkyl or by both;wherein each of R₄, R₅, R₆ and R₈ is independently selected from H orlower alkyl; wherein R₇ is selected from substituted or unsubstitutedcycloalkyl, phenyl, cycloalkylalkyl and phenylalkyl, any one of whichmay be substituted with one or more groups selected from alkyl, alkoxy,halo, haloalkyl, lower alkenyl, lower alkynyl and cyano; and whereineach of R₁₀ and R₁₁ is independently selected from the groups H, loweralkyl, cycloalkyl, phenyl, benzyl, naphthyl and naphthylmethyl, any oneof which groups having a substitutable position may be optionallysubstituted with or more of lower alkyl, alkoxy, alkenyl, alkynyl, halo,haloalkyl, cyano and phenyl, with the proviso that at least one of R₁₀and R₁₁ is an aryl group; or a pharmacologically-acceptable saltthereof.
 16. The method of claim 15 wherein said renin-inhibitingcompound is of the formula ##STR25## wherein R₁ through R₁₁ are asdefined before; or a pharmacologically-acceptable salt thereof.
 17. Themethod of claim 16 wherein said compound isBoc-L-phenylalaninyl-D,L-α-methyl-β-alaninamide of(2S,3R,4S)-2-amino-1-cyclohexyl-3,4-dihydroxy-6-methylheptane; or apharmacologically-acceptable salt thereof.
 18. The method of claim 16wherein said compound is Boc-L-phenylalaninyl-D,L-α-methyl-β-alaninamideof (2S,3R,4S)-2-amino-1-cyclohexyl-3,4-dihydroxypentane; or apharmacologically-acceptable salt thereof.
 19. The method of claim 16wherein said compound is Boc-L-phenylalaninyl-D,L-α-methyl-β-alaninamideof (2S,3R)-2-amino-1-cyclohexyl-3,4-dihydroxybutane; or apharmacologically-acceptable salt thereof.
 20. The method of claim 16wherein R₉ is ##STR26## wherein R₁ is selected from H, linear orbranched lower alkyl, haloalkyl, alkylcycloalkyl, alkylcycloalkenyl andalkoxycarbonyl; wherein R₂ is selected from linear or branched loweralkyl, imidazomethyl and benzyl; wherein R₃ is selected from loweralkyl, acylaminoalkyl, benzyl, naphthylmethyl, aryl and benzylsubstituted at the phenyl portion by halo or lower alkyl or by both;wherein each of R₄, R₅, R₆ and R₈ is independently selected from H orlower alkyl; wherein R₇ is selected from substituted or unsubstitutedcycloalkyl, phenyl, cycloalkylalkyl and phenylalkyl, any one of whichmay be substituted with one or more groups selected from alkyl, alkoxy,halo, haloalkyl, lower alkenyl, lower alkynyl and cyano; wherein each orR₁₀ and R₁₁ is independently selected from the groups H, lower alkyl,cycloalkyl, phenyl, benzyl, naphthyl, and naphthylmethyl, any one ofwhich groups having a substitutable position may be optionallysubstituted with or more of lower alkyl, alkoxy, alkenyl, alkynyl, halo,haloalkyl, cyano and phenyl, with the proviso that at least one of R₁₀and R₁₁ is an aryl group; or a pharmacologically-acceptable saltthereof.
 21. The method of claim 20 wherein R₁ is isobutyl; R₂ isisobutyl; R₃ is benzyl; R₄ is H or methyl; R₅ is H or methyl; R₆ is H ormethyl; R₇ is cyclohexylmethyl; R₈ is H or methyl; each of R₁₀ and R₁₁is independently selected from H, lower alkyl and phenyl, with at leastone of R₁₀ and R₁₁ being phenyl; or a pharmacologically-acceptable saltthereof.